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Uptake of sulfur-35-labeled carbon disulfide by Ascaridia galli (roundworm) and its chicken host
EXPERIMENTAL
1’ARASITOLOGY
Uptake
9,
56-62 (1960)
of Sulfur-35-Labeled (Roundworm) S. E. Knapp,”
M. F. Hansen,”
Carbon Disulfide by Ascaridia and Its Chicken Host”2 H. C. Moser,4
(Submit tctl for l)ubli~;ction,
and
13 February
galli
R. H. McFarland5
1’359)
1. IPLP&W c~xperiments using Ascrrritlin grtlli and S’“-labeled c.arhon disulfide indicated that CH,“” was taken u*, by the worm itS a v;lpor. Wlicn uptake of tll~ug was c~ompirrd with ligatrd and nonligat,cd worms, no differenc*rs could he drmonst~~atcd, indic*uting that. CS, rntcred the worm via t,he cut.klc. 2. Exrluding size diffcl,enc.c,s inhr,rc,nt hctwcrn malt ;md frmalc worms, iu l&ro or irl l~iz~j tests showed that WY did not influence drug uptake. There was an indication that the number of pg of drug per worm was greater for smallrr worms than for larger ones, hut not enough worms were tcstrd to establish this point statistically. 3. As cxposurc time to CH,“” was infwwsed geomctri~~tlly xt. a f*onstnnt dosage,, :L sat,uration point was q)l)roximntcd for female :~s~:tritls treated in I&W. This was nlso t,rue when dosage WBS inwxncd geomrt Gcally and exposure timr was kept c*onst;tnt,. In both f’ases the sni uriltion point was near 3.0 pg per mg of worm. 4. Individual worm analysis revealctl that the body fluid took ~1) the greatest quantity of Y:‘” ectuivalpnts of CS,” l)cr mg of tissue. Body fluid was followed, in rcspcrt to drug II~Itake, hy the reproduct,ivc xystcm, intestinc, and body w~dl, rrsl)cctivrly. 5. 1,b viva studies using the &mot hcr:rpcntic dose of CS,“” sliowvrfl that. St’ f~cpi\-:ilf9ts of C81”” Lvere prcscnt in 22 tissurs and organs of the rhickcn at 48 hogs I)ost-tI,c~ttmrnt. The> highest concentration of V’ was found in the gall bladder and the lowest in the bmin. One hundred per cent efficG*y was attained in ~c11 of the three c,hicakr,ns treated. The cxpellcd worms contained im ;fveritge of 0.087 pg of 8:‘” prr tng. 6. The rhickcn escreta, bot,h urinal and feces, had an average \,:~lur of 0.245 pg of S:‘” per mg. There was no difference in drlig lcvcl in excrcta containing worms or without. worms.
Only a fcm reports have been published concerning the use of a radioactively labeled anthclmintic for studying its mode of action on a parasite and its host (Brady et al., 1945; Lawton et al., 1945; Lazarus and Rogers, 1951; Esscman, 1952; and Tcrllaar, 1957). These studies have revealed valuable information, otherwise lacking or difficult to obtain, concerning the antlicl-
nlintic activity of the particular drug under investigation. Although CS, is principally used to rcnlovc stonmcll hots and certain nematode parasitts from horses and swine, the test l,arasitc in the prcscnt study was ilscaridia ynlli and its chicken host. The purpose of the study was to dctcrmine by in vitro and in viva cxpcriments tlic niode of action of sulfur-35 labeled carbon disulfide (CS,:in) with respect to the rate of penetration, mode of entry, and distribution of the drug in tllc parasite and its host.
‘Cont.ribution
No. 288, 580, and 70, three deIGinsns Agric~ulturnl Exprrimmt Stat,ion, Manhattan, Kansas. prescntrd Iy S. E. ‘A portion of ix dissertation Knapp in partial fulfillment of tllc recluirctncnts for the degree Dort.or of Philoso~~hy in Parasitology at. Kansas State College of Agrirultrirc, antI L%l)plicd S:.irnt.r, M:mhatt:m. ” Depnrtment, of Zoology. ’ Df,1):kt,t tnfat of Chemist 1.y. ’ Deputnient of Physi(~. p:wtmcnts,
respectively;
S!fnthesis
and
I)eterusination
of
CS,z{z
Sulfur-35labeled CS, n-as synthesized using a modifiration of the nwthod dcacribcd by Edwards et nl. ( 1948 1. Elcnwntal 56
S35-~~~~~~~ CS2 UPTAKE BY ROUNDWORMS AND HOSTS
sulfur-35 dissolved in benzene was pipetted, in 100 ~1 quantities, into a 73 X 11-mm Pyrex test tube containing approximately 25 mg of sulfur. The tube was then heated and a fine jet of air was passed over its open end to remove the benzene. After drying, the W mixture was covered with about a 2-cm layer of iron filings and heat’ed until most of the sulfur reacted with the iron, forming FeS35. The FeW was transferred to a round-bottomed, side-arm reaction flask. To produce H&Y, a 1:l solution of concentrated HCl and water was slowly dripped over the FeS”” from a separation funnel attached to the reaction flask. The HaS3s formed was carried from the flask by a steady stream of helium. The helium stream was passed through the flask and into 10 ml of 0.1 N NaOH for 45 minutes. The S”“-labeled Na&Y formed in the NaOH solution was then added to 10 ml of redistilled CSz contained in a 50-ml flask and mixed with the aid of a magnetic stirring device for eight hours at room temperature. By exchange, CSZ3” was produced along with unaltered CS2. The labeled and unlabeled CS, was purified by redistillation and had a specific activity of 0.8 millicuries per milliliter. Radioassays of the labeled CS, and the experimental samples were made as follows. The specific activity of the CS,3s was determined by transferring 10 ml to a 25 ml volumetric flask containing ethyl alcohol and a known quantity (ca. 860 mg) of reagent grade carbon disulfide. The flask was filled to volume with ethyl alcohol and mixed. One hundred ~1 were transferred to a gelatin capsule and oxidized in a 22 ml Parr Bomb (Parr, 1919). This sample contained approximately 3.5 mg of C& and yielded 20 mg of BaS04 when precipitated with BaC12. For tissue or worm samples, 2 ml of a solution containing 7.47 mg KZSOI per ml was added as carrier for the radioactive sulfur to provide a final BaS04 precipitate similar to that of the CS235specific activity standard. The sulfate resulting from the Parr Bomb oxidation was separated prior to precipitation with BaClz by running the solution through anion and cation exchange columns in a manner simi-
57
lar to that described by Fritz and Hammond (1957). Counting rates for all S35 samples were determined using a Geiger-Miiller counter with an end window thickness of 1.92 mg/crn” in conjunction with a Berkeley Decimal Scaler, Model 2105. Corrections were made for background and sample decay according to standard procedures. Geometry was approximately 10%. In Vitro Experiments Freshly recovered Ascaridia galli were washed and stored in Locke’s solution at 41°C. None of the worms used in these tests was outside of the host more than 12 hours. Worms were individually exposed to the vapors of CS,35 by suspending them in a closed glass tube (volume ca. 85 ml) supported in a water bath at 41°C. At this temperature, equivalent to that of the chicken, CS, is rapidly vaporized. In testing the mode of entry of the drug, a number of male worms were ligated (below the mouth and above the anus) with a fine silk thread. Each was exposed for 5 minutes to 50 ~1 of the drug. A second group of nonligated male worms was exposed similarly. Also, nonligated female worms were treated in order to provide a comparison between sexes as to the uptake of the drug. In addition, females of different sizes were treated in order to determine the relationship between uptake of drug and size of worms. The rate of uptake of CS# was determined by exposing a series of worms t’o different concentrations of the drug for a constant period of time and by exposing worms to constant quantities of the drug for varying times. Several worms were dissected after exposure to CSid8 and their individual parts analyzed for total S35 content. The intestinal tract, reproductive system, and body wall were separately removed, dried, weighed and analyzed. Also, perienteric fluid was removed by applying a weighed glass capillary tube to an incision on the body wall. After removal of this fluid, the tube was reweighed and oxidized in the Parr Bomb, prior to analysis.
58
KNAPP,
HANSEN,
MOSER AND MCFARLAND
In Vivo Experiments The purpose of these experiments was to determine affinities of CS2 for various tissues and organs of the host and to compare the uptake of these with that of the parasites. Three female White Rock chickens, infected with mature Ascaridia galli, were treated with 0.35 ml of CS335per kilogram (Knapp and Hansen, 1954). Each bird was fasted for 15 hours and then given the drug in a gelatin capsule introduced directly into the crop. Blood samples were removed periodically from the wing and analyzed for S35. Randomly selected urine and fecal samples were also examined for S3j content. As the parasites were passed, they were collected, rinsed in Locke’s solution and analyzed. Forty-eight hours posttreatment, the birds were autopsied and portions of tissues and organs were preserved by quick-freezing for later S35analyses. In tests involving the alimentary tract, t#he thyme was rinsed from the tissues prior to analysis. The sulfur-35 contents of these samples were measured. Since there was no way of knowing whether the material counted represented the drug or a metabolite of the drug, the counting measurements were used to calculate the amount of sulfur (from CS235) required to give these counting rates. These quantities are referred to as S35 equivalents of CS235 or simply as micrograms of S35.
TABLE II Worm Size in Relation to Sorption Worm No. 1 2 3 4 5 6 7 8 9 10 Mean * In
Weight (mg) 34.0 44.7 65.8 70.2 74.1 75.6 97.3 100.0 101.2 163.2 82.61
vitrotest;
of CSf*
Pg of cs3s 2 per worm
pg of c@ per mg of worm
54 57 65 69 73 80 84 74 83 95
80.9 80.1 110.8 117.2 141.5 99.4 152.5 193.1 116.9 148.7
2.38 1.79 1.68 1.67 1.43 1.32 1.55 1.93 1.15 0.91
73
124.1
1.58
Length (mm)
female ascarids, 50 ~1 dose, 5-minute expmwe.
ized CS,“” penetrated the cuticle of the worms since there was no significant difference in drug uptake between the ligated and nonligated groups. The significant differences between total uptake of nonligated females and males is related to the larger size of the females (Table I). The statistical test (Tang, 1938) was powerful enough to detect a difference of 0.364 pg of CSZ3” per mg between the two groups. These in vitro experiments provided support for the hypothesis that the cuticle was the site of entry of the drug into the worm. Lazarus and Rogers (1951) have indicated that the cuticle of Ascaridia galli is likewise the site of penetration of phenothiazine. RESULTS AND DIMXJSSION A series of nonligated female worms was exposed to the drug to determine the In Vitro Experiments amount taken up by different sized worms Worms exposed to the vapor of CS235 (Table II). An analysis of the data indiwere analyzed for total CS235 as well as cated that as the total worm weight infor pg of drug per mg of worm. In these creased the total pg of anthelmintic inexperiments, the worms were killed by the creased. Mean values for the experiment vapors of CS235. It is assumed that vaporrevealed that the sorption of drug for a TABLE I worm weighing 82.61 mg would be 124.11 pg and that this would be distributed over Uptake qf CS:” bv Ascaridia galli Treated in Vitro the worm at a concentration of 1.58 pg per pg of csi5/ pg of cst5/ mg. There was, however, an indication that Treatment mg of worm worm as worm weight increased, the pg of CS235 per mg of worm decreased but the relation1.33 50.1 Ligated males ship was not significant for the degrees of 1.60 64.4 Nonligated males freedom involved. 1.58 124.1 Nonligated females A study was made on the influence of ex-
a50
t
I
I
I
I
I
2
4
8
16
TIME (Min.) between exposure time and uptake of CSP for female Ascaridiu FIG. 1. Relationship galli. Three worms exposed to 50 ~1 doses for each time interval. Mean values are shown.
I
I 25
I 50
II
II
200 200
ID0 100 MICROLITERS Ce
ADMINISTERED
FIG. 2. Relationship between dosage and uptake of C&” for female Ascaridiu Three worms exposed for 5 minutes for each dosage. Mean values are shown. 59
galli.
60
KNAPP,
HANSEN,
MOSER
posure time and dosage on the uptake of CS2a6by female ascarids. Each worm was exposed in a closed system at 41°C to the vapors of 50 ~1 of CS235for 1, 2, 4, 8, and 16 minutes, respectively. The mean values in pg of CS235per mg of worm are shown in Fig. 1. An analysis of the data indicated that the relationship between the length of exposure and the uptake of CS235was curvilinear. Since a quadratic curve best fits these data, it may be stated that the concentration of C&a5 tended to level off as exposure time was increased. There was no evidence that maximum saturation was reached in this experiment. Another series of experiments used a constant exposure time but varied the dosage of CS235.Worms were exposed for five minutes to volumes of 25, 50, 100, and 200 pl of CS235,respectively (Fig. 2). Since the size variance among worms within the same treatment group was unusually large in this experiment, it was difficult to determine the best fitting line. A linear line will fit the data but there was evidence from the analysis of variance that a quadratic line would again be more descriptive. However, this was inconclusive (.05 < P < .lO) and additional evidence will be required before one can postulate a curvilinear line at the minimum level of probability (.05) used throughout this study. A comparison of the results from these two experiments indicated that the number of pg of CSZs6per mg of worm was quite similar as dosage LllCROGRAYS OF SULFUR-35 EQUIVALENTS OF CS, PER GRAM OF TISSUE
FIQ. 3. Uptake of S% equivalents of CSzm by the various body parts of adult female ascarids exposed in vitro to the vapors of the drug. Mean values are represented.
AND
MCFARLAND
or exposure time was increased. Both curves are descriptive of the test system used. Considering the average uptake of S3j equivalents of CS235 by Ascaridia galli treated in ‘uiz~o,it is possible that one could obtain comparable results in vitro if either the exposure time or volume of drug administered was reduced. Perhaps by considering both the dosage level and time of exposure in in vitro experiments, an LDjo value could be determined. An in vitro study was made on the distribution of S35 equivalents of CS236in Ascaridia galli. The body wall, intestine, reproductive organs and body fluid were removed for analysis. Concentration of the drug was measured in /*g of S35per mg of worm, thus the effects of the weights of individual worms did not bias the results (Fig. 3). A comparison of the mean values by Duncan’s Multiple Range Test (1955) revealed that the average amounts of S35 equivalents of CS235taken up by the body wall, intestine, and reproductive system were not significantly different, whereas, the body fluid contained a significantly greater amount (P < .05) of S35.Speculation as to the presence of relatively high concentrations of S35 in the body fluid suggests: 1) that it accumulated as waste material prior to its elimination and that it had already rendered its toxic effect on the worm, or 2) that the body fluid was, in part, the site of anthelmintic activity. In Vivo Experiments An analysis of host blood samples showed that S35was present 15 minutes after administration of the drug and persisted until the birds were killed 48 hours post-treatment (Table III). The highest mean concentration of S35 equivalents of CS235in the blood occurred 16 hours after treatment. Since blood samples were not removed at int’ervals between the times given in Table III, there was no way of knowing whether the 16-hour sample represented maximum blood saturation with the anthelmintic. At 48 hours post-treatment, 0.024 pg of S35 per mg of blood was detected. Of the tissues and/or organs tested, the greatest concentration of S35equivalents of CS235 was in the gall bladder, and the
CS2 UPTAKE BY ROUNDWORMS AND HOSTS
S35-~~~~~~ TABLE
61
III
Micrograms of Sulfur-35 per Milligram of Blood for Samples Removed at Various Time Intervals from Chickens Treated with 0.55 ml CS:” per Kilogram Body-weight
Blood sample (hours) ControP 0.25 0.50 1.00 2.00 4.00 8.00 16.00 32.00 48.00
Chicken 1
2
0.013 0.016 0.020 0.025 0.023 0.030 0.035 0.027 0.016
0.011 0.021 0.022 0.033 0.035 0.032 0.038 0.035 0.023
-
D A blood aample was removed from ment a.a a check for natural radioactivity. b This sample WBB lost in one of the ures.
3
s
0.000
0.000
0.008 -b 0.022 0.024 0.026 0.029 0.035 0.040 0.033
0.011 0.018 0.021 0.027 0.028 0.030 0.036 0.034 0.024
bird
(3) prior
concentration
to treatproced-
smallest concentration in the brain (Fig. 4). Nine tissues and/or organs had quantities of S35 within 0.006 rg per mg of the 48-hour blood sample. Table IV gives the mean uptake of S3” equivalents of CS,35 by 11 male and 14 female worms expelled from the treated birds 24 hours post-treatment. The mean amount of EY5recorded per worm was 0.087 pg per mg. Mean uptake of S35 for male and female worms was 0.1047 and 0.0821 (not significant) rg per mg, respectively. The amount of S35 per mg of excreta (urine and feces) is given in Table V. These data represent pg per mg of S35 for randomly selected samples rather than for each elimination, since many were passed within a few minutes of each other. The average yield of S35 per mg of excreta for all three chickens was 0.245 pg. The amount was two to three times greater than that recorded for the worms. Also, excreta containing worms was no different in S35 content, from excreta without worms. Since the fecal and urine components of the excreta were not separated prior to analysis, the fraction of S3j present in each could not be ascertained. McKee et al. (1943) indicated that in mammals 0.5% of the retained
cBRAIN FIG. 4. Comparison of S” equivalents of CSZW contained by Ascario!ia galli and certain tissues and organs of the host after treatment with the drug. Mean values are represented. (Gall bladder represents a cross section of frozen gall bladder and its contents.)
TABLE
IV
Mean ~9%Equivalents of CS:’ for Ascarids Expelled by Treated Chickens
Bird No.
Number worms
pg W per worm
pg W per mg of worm
7 12 6
8.01 10.15 8.75
0.076 0.091 0.093
1
2 3
TABLE
V
Mean S3’ Equivalents of CS? for Selected Sample of Excreta from Treated Chickens Bird
No. 1
2 3
pg W per mg of excreta 0.273 0.249 0.213
KNAPP, HANSEN, MOSER AND MCFARLAND
62
CSz3” occurred in the urine and none in the feces and that the remaining CS2 was accounted for as metabolites excreted in the urine. ACKNOWLEDGMENTS The authors wish to acknowledge the assistance of Dr. Stanley Wearden, Kansas State College Statistical Laboratory, and appreciation to the United States Atomic Energy Commission for funds supporting the study (Contract AT(ll-l)-
308). REFERENCES BRADY, F. J., LAWTON, A. H., COWIE, D. B., ANDREWS,H. L., NESS, A. T., ANDOGDEN,GLEN E. 1945. Localization of trivalent radioactive antimony following intravenous administration to dogs infected with Dirojiluria immitis. Am. J. Trop. Med. 25(2),
103-107.
DUNCAN, D. B. 1955. Multiple range and muItiple F tests. Biometrics 11, 142. EDWARDS,R. R., NESBETT,F. B., ANDSOLOMON, A. K. 1948. Recoil-activated and thermal exchange reactions between sulfur-35 and carbon disulfide. J. Am. Chem. Sot. 70,167O. ESSERMAN,H. B. 1952. The mode of action of phenothiazine as an anthelmintic. II. Phenothiazine in the intestinal fluid and nematode parasites of treated animals. Australian J. Sci. Research
B5(4), 485495.
FRITZ, J. S., ANDHAMMOND,G. S. 1957. Quantitative
Organic Analysis. John Wiley and Sons, Inc., New York, 303 pp. KNAPP, S. E., AND HANSEN, M. F. 1954. Observations on the anthelmintic action of carbon disulfide on the fowl ascarid Ascaridia galli (Schrank). J. Parasitol. 40, 17-18. LAWTON,A. H., NESS, A. T., BRADY,F. J., ANDDEAN, B. C. 1945. Distribution of radioactive arsenic following intraperitoneal injection of sodium arsenit.e into cotton rats infected with Litomosoides carinii. Science 102,121X122. LAZARUS,M. AND ROGERS,W. P. 1951. The mode of action of phenothiaeine as an anthelmintic. The uptake of ?S-labelled phenothiazine by the tissues of nematode parasites and their hosts. Australian J. Sci. Research B4(2), 163179. MCKEE, R. W., KIPER, C., FOUNTAIN,J. H., RISKIN, A. M., ANDDRINKER, P. 1943. A solvent vapor, carbon disulfide : Absorption, elimination, metabolism and mode of action. J. Am. Med. Assoc. 122,217-222.
PARR, S. W. 1919. A fusion bomb for sulfur determination in coal. J. Znd. Eng. Chem. 11,
230. TANG, P. C. 1938. The power function of the analysis of variance tests with tables and illustrations of their use. Stat. Research Mem. 2, 126157. TERHAAR, C. J. 1957. Carbon tetrachloride as an anthelmintic: Its effect on and distribution in a parasite (Ascaridia galli) and its host. Unpublished Dissertation, Kansas State College, 97 p.
1’ARASITOLOGY
Uptake
9,
56-62 (1960)
of Sulfur-35-Labeled (Roundworm) S. E. Knapp,”
M. F. Hansen,”
Carbon Disulfide by Ascaridia and Its Chicken Host”2 H. C. Moser,4
(Submit tctl for l)ubli~;ction,
and
13 February
galli
R. H. McFarland5
1’359)
1. IPLP&W c~xperiments using Ascrrritlin grtlli and S’“-labeled c.arhon disulfide indicated that CH,“” was taken u*, by the worm itS a v;lpor. Wlicn uptake of tll~ug was c~ompirrd with ligatrd and nonligat,cd worms, no differenc*rs could he drmonst~~atcd, indic*uting that. CS, rntcred the worm via t,he cut.klc. 2. Exrluding size diffcl,enc.c,s inhr,rc,nt hctwcrn malt ;md frmalc worms, iu l&ro or irl l~iz~j tests showed that WY did not influence drug uptake. There was an indication that the number of pg of drug per worm was greater for smallrr worms than for larger ones, hut not enough worms were tcstrd to establish this point statistically. 3. As cxposurc time to CH,“” was infwwsed geomctri~~tlly xt. a f*onstnnt dosage,, :L sat,uration point was q)l)roximntcd for female :~s~:tritls treated in I&W. This was nlso t,rue when dosage WBS inwxncd geomrt Gcally and exposure timr was kept c*onst;tnt,. In both f’ases the sni uriltion point was near 3.0 pg per mg of worm. 4. Individual worm analysis revealctl that the body fluid took ~1) the greatest quantity of Y:‘” ectuivalpnts of CS,” l)cr mg of tissue. Body fluid was followed, in rcspcrt to drug II~Itake, hy the reproduct,ivc xystcm, intestinc, and body w~dl, rrsl)cctivrly. 5. 1,b viva studies using the &mot hcr:rpcntic dose of CS,“” sliowvrfl that. St’ f~cpi\-:ilf9ts of C81”” Lvere prcscnt in 22 tissurs and organs of the rhickcn at 48 hogs I)ost-tI,c~ttmrnt. The> highest concentration of V’ was found in the gall bladder and the lowest in the bmin. One hundred per cent efficG*y was attained in ~c11 of the three c,hicakr,ns treated. The cxpellcd worms contained im ;fveritge of 0.087 pg of 8:‘” prr tng. 6. The rhickcn escreta, bot,h urinal and feces, had an average \,:~lur of 0.245 pg of S:‘” per mg. There was no difference in drlig lcvcl in excrcta containing worms or without. worms.
Only a fcm reports have been published concerning the use of a radioactively labeled anthclmintic for studying its mode of action on a parasite and its host (Brady et al., 1945; Lawton et al., 1945; Lazarus and Rogers, 1951; Esscman, 1952; and Tcrllaar, 1957). These studies have revealed valuable information, otherwise lacking or difficult to obtain, concerning the antlicl-
nlintic activity of the particular drug under investigation. Although CS, is principally used to rcnlovc stonmcll hots and certain nematode parasitts from horses and swine, the test l,arasitc in the prcscnt study was ilscaridia ynlli and its chicken host. The purpose of the study was to dctcrmine by in vitro and in viva cxpcriments tlic niode of action of sulfur-35 labeled carbon disulfide (CS,:in) with respect to the rate of penetration, mode of entry, and distribution of the drug in tllc parasite and its host.
‘Cont.ribution
No. 288, 580, and 70, three deIGinsns Agric~ulturnl Exprrimmt Stat,ion, Manhattan, Kansas. prescntrd Iy S. E. ‘A portion of ix dissertation Knapp in partial fulfillment of tllc recluirctncnts for the degree Dort.or of Philoso~~hy in Parasitology at. Kansas State College of Agrirultrirc, antI L%l)plicd S:.irnt.r, M:mhatt:m. ” Depnrtment, of Zoology. ’ Df,1):kt,t tnfat of Chemist 1.y. ’ Deputnient of Physi(~. p:wtmcnts,
respectively;
S!fnthesis
and
I)eterusination
of
CS,z{z
Sulfur-35labeled CS, n-as synthesized using a modifiration of the nwthod dcacribcd by Edwards et nl. ( 1948 1. Elcnwntal 56
S35-~~~~~~~ CS2 UPTAKE BY ROUNDWORMS AND HOSTS
sulfur-35 dissolved in benzene was pipetted, in 100 ~1 quantities, into a 73 X 11-mm Pyrex test tube containing approximately 25 mg of sulfur. The tube was then heated and a fine jet of air was passed over its open end to remove the benzene. After drying, the W mixture was covered with about a 2-cm layer of iron filings and heat’ed until most of the sulfur reacted with the iron, forming FeS35. The FeW was transferred to a round-bottomed, side-arm reaction flask. To produce H&Y, a 1:l solution of concentrated HCl and water was slowly dripped over the FeS”” from a separation funnel attached to the reaction flask. The HaS3s formed was carried from the flask by a steady stream of helium. The helium stream was passed through the flask and into 10 ml of 0.1 N NaOH for 45 minutes. The S”“-labeled Na&Y formed in the NaOH solution was then added to 10 ml of redistilled CSz contained in a 50-ml flask and mixed with the aid of a magnetic stirring device for eight hours at room temperature. By exchange, CSZ3” was produced along with unaltered CS2. The labeled and unlabeled CS, was purified by redistillation and had a specific activity of 0.8 millicuries per milliliter. Radioassays of the labeled CS, and the experimental samples were made as follows. The specific activity of the CS,3s was determined by transferring 10 ml to a 25 ml volumetric flask containing ethyl alcohol and a known quantity (ca. 860 mg) of reagent grade carbon disulfide. The flask was filled to volume with ethyl alcohol and mixed. One hundred ~1 were transferred to a gelatin capsule and oxidized in a 22 ml Parr Bomb (Parr, 1919). This sample contained approximately 3.5 mg of C& and yielded 20 mg of BaS04 when precipitated with BaC12. For tissue or worm samples, 2 ml of a solution containing 7.47 mg KZSOI per ml was added as carrier for the radioactive sulfur to provide a final BaS04 precipitate similar to that of the CS235specific activity standard. The sulfate resulting from the Parr Bomb oxidation was separated prior to precipitation with BaClz by running the solution through anion and cation exchange columns in a manner simi-
57
lar to that described by Fritz and Hammond (1957). Counting rates for all S35 samples were determined using a Geiger-Miiller counter with an end window thickness of 1.92 mg/crn” in conjunction with a Berkeley Decimal Scaler, Model 2105. Corrections were made for background and sample decay according to standard procedures. Geometry was approximately 10%. In Vitro Experiments Freshly recovered Ascaridia galli were washed and stored in Locke’s solution at 41°C. None of the worms used in these tests was outside of the host more than 12 hours. Worms were individually exposed to the vapors of CS,35 by suspending them in a closed glass tube (volume ca. 85 ml) supported in a water bath at 41°C. At this temperature, equivalent to that of the chicken, CS, is rapidly vaporized. In testing the mode of entry of the drug, a number of male worms were ligated (below the mouth and above the anus) with a fine silk thread. Each was exposed for 5 minutes to 50 ~1 of the drug. A second group of nonligated male worms was exposed similarly. Also, nonligated female worms were treated in order to provide a comparison between sexes as to the uptake of the drug. In addition, females of different sizes were treated in order to determine the relationship between uptake of drug and size of worms. The rate of uptake of CS# was determined by exposing a series of worms t’o different concentrations of the drug for a constant period of time and by exposing worms to constant quantities of the drug for varying times. Several worms were dissected after exposure to CSid8 and their individual parts analyzed for total S35 content. The intestinal tract, reproductive system, and body wall were separately removed, dried, weighed and analyzed. Also, perienteric fluid was removed by applying a weighed glass capillary tube to an incision on the body wall. After removal of this fluid, the tube was reweighed and oxidized in the Parr Bomb, prior to analysis.
58
KNAPP,
HANSEN,
MOSER AND MCFARLAND
In Vivo Experiments The purpose of these experiments was to determine affinities of CS2 for various tissues and organs of the host and to compare the uptake of these with that of the parasites. Three female White Rock chickens, infected with mature Ascaridia galli, were treated with 0.35 ml of CS335per kilogram (Knapp and Hansen, 1954). Each bird was fasted for 15 hours and then given the drug in a gelatin capsule introduced directly into the crop. Blood samples were removed periodically from the wing and analyzed for S35. Randomly selected urine and fecal samples were also examined for S3j content. As the parasites were passed, they were collected, rinsed in Locke’s solution and analyzed. Forty-eight hours posttreatment, the birds were autopsied and portions of tissues and organs were preserved by quick-freezing for later S35analyses. In tests involving the alimentary tract, t#he thyme was rinsed from the tissues prior to analysis. The sulfur-35 contents of these samples were measured. Since there was no way of knowing whether the material counted represented the drug or a metabolite of the drug, the counting measurements were used to calculate the amount of sulfur (from CS235) required to give these counting rates. These quantities are referred to as S35 equivalents of CS235 or simply as micrograms of S35.
TABLE II Worm Size in Relation to Sorption Worm No. 1 2 3 4 5 6 7 8 9 10 Mean * In
Weight (mg) 34.0 44.7 65.8 70.2 74.1 75.6 97.3 100.0 101.2 163.2 82.61
vitrotest;
of CSf*
Pg of cs3s 2 per worm
pg of c@ per mg of worm
54 57 65 69 73 80 84 74 83 95
80.9 80.1 110.8 117.2 141.5 99.4 152.5 193.1 116.9 148.7
2.38 1.79 1.68 1.67 1.43 1.32 1.55 1.93 1.15 0.91
73
124.1
1.58
Length (mm)
female ascarids, 50 ~1 dose, 5-minute expmwe.
ized CS,“” penetrated the cuticle of the worms since there was no significant difference in drug uptake between the ligated and nonligated groups. The significant differences between total uptake of nonligated females and males is related to the larger size of the females (Table I). The statistical test (Tang, 1938) was powerful enough to detect a difference of 0.364 pg of CSZ3” per mg between the two groups. These in vitro experiments provided support for the hypothesis that the cuticle was the site of entry of the drug into the worm. Lazarus and Rogers (1951) have indicated that the cuticle of Ascaridia galli is likewise the site of penetration of phenothiazine. RESULTS AND DIMXJSSION A series of nonligated female worms was exposed to the drug to determine the In Vitro Experiments amount taken up by different sized worms Worms exposed to the vapor of CS235 (Table II). An analysis of the data indiwere analyzed for total CS235 as well as cated that as the total worm weight infor pg of drug per mg of worm. In these creased the total pg of anthelmintic inexperiments, the worms were killed by the creased. Mean values for the experiment vapors of CS235. It is assumed that vaporrevealed that the sorption of drug for a TABLE I worm weighing 82.61 mg would be 124.11 pg and that this would be distributed over Uptake qf CS:” bv Ascaridia galli Treated in Vitro the worm at a concentration of 1.58 pg per pg of csi5/ pg of cst5/ mg. There was, however, an indication that Treatment mg of worm worm as worm weight increased, the pg of CS235 per mg of worm decreased but the relation1.33 50.1 Ligated males ship was not significant for the degrees of 1.60 64.4 Nonligated males freedom involved. 1.58 124.1 Nonligated females A study was made on the influence of ex-
a50
t
I
I
I
I
I
2
4
8
16
TIME (Min.) between exposure time and uptake of CSP for female Ascaridiu FIG. 1. Relationship galli. Three worms exposed to 50 ~1 doses for each time interval. Mean values are shown.
I
I 25
I 50
II
II
200 200
ID0 100 MICROLITERS Ce
ADMINISTERED
FIG. 2. Relationship between dosage and uptake of C&” for female Ascaridiu Three worms exposed for 5 minutes for each dosage. Mean values are shown. 59
galli.
60
KNAPP,
HANSEN,
MOSER
posure time and dosage on the uptake of CS2a6by female ascarids. Each worm was exposed in a closed system at 41°C to the vapors of 50 ~1 of CS235for 1, 2, 4, 8, and 16 minutes, respectively. The mean values in pg of CS235per mg of worm are shown in Fig. 1. An analysis of the data indicated that the relationship between the length of exposure and the uptake of CS235was curvilinear. Since a quadratic curve best fits these data, it may be stated that the concentration of C&a5 tended to level off as exposure time was increased. There was no evidence that maximum saturation was reached in this experiment. Another series of experiments used a constant exposure time but varied the dosage of CS235.Worms were exposed for five minutes to volumes of 25, 50, 100, and 200 pl of CS235,respectively (Fig. 2). Since the size variance among worms within the same treatment group was unusually large in this experiment, it was difficult to determine the best fitting line. A linear line will fit the data but there was evidence from the analysis of variance that a quadratic line would again be more descriptive. However, this was inconclusive (.05 < P < .lO) and additional evidence will be required before one can postulate a curvilinear line at the minimum level of probability (.05) used throughout this study. A comparison of the results from these two experiments indicated that the number of pg of CSZs6per mg of worm was quite similar as dosage LllCROGRAYS OF SULFUR-35 EQUIVALENTS OF CS, PER GRAM OF TISSUE
FIQ. 3. Uptake of S% equivalents of CSzm by the various body parts of adult female ascarids exposed in vitro to the vapors of the drug. Mean values are represented.
AND
MCFARLAND
or exposure time was increased. Both curves are descriptive of the test system used. Considering the average uptake of S3j equivalents of CS235 by Ascaridia galli treated in ‘uiz~o,it is possible that one could obtain comparable results in vitro if either the exposure time or volume of drug administered was reduced. Perhaps by considering both the dosage level and time of exposure in in vitro experiments, an LDjo value could be determined. An in vitro study was made on the distribution of S35 equivalents of CS236in Ascaridia galli. The body wall, intestine, reproductive organs and body fluid were removed for analysis. Concentration of the drug was measured in /*g of S35per mg of worm, thus the effects of the weights of individual worms did not bias the results (Fig. 3). A comparison of the mean values by Duncan’s Multiple Range Test (1955) revealed that the average amounts of S35 equivalents of CS235taken up by the body wall, intestine, and reproductive system were not significantly different, whereas, the body fluid contained a significantly greater amount (P < .05) of S35.Speculation as to the presence of relatively high concentrations of S35 in the body fluid suggests: 1) that it accumulated as waste material prior to its elimination and that it had already rendered its toxic effect on the worm, or 2) that the body fluid was, in part, the site of anthelmintic activity. In Vivo Experiments An analysis of host blood samples showed that S35was present 15 minutes after administration of the drug and persisted until the birds were killed 48 hours post-treatment (Table III). The highest mean concentration of S35 equivalents of CS235in the blood occurred 16 hours after treatment. Since blood samples were not removed at int’ervals between the times given in Table III, there was no way of knowing whether the 16-hour sample represented maximum blood saturation with the anthelmintic. At 48 hours post-treatment, 0.024 pg of S35 per mg of blood was detected. Of the tissues and/or organs tested, the greatest concentration of S35equivalents of CS235 was in the gall bladder, and the
CS2 UPTAKE BY ROUNDWORMS AND HOSTS
S35-~~~~~~ TABLE
61
III
Micrograms of Sulfur-35 per Milligram of Blood for Samples Removed at Various Time Intervals from Chickens Treated with 0.55 ml CS:” per Kilogram Body-weight
Blood sample (hours) ControP 0.25 0.50 1.00 2.00 4.00 8.00 16.00 32.00 48.00
Chicken 1
2
0.013 0.016 0.020 0.025 0.023 0.030 0.035 0.027 0.016
0.011 0.021 0.022 0.033 0.035 0.032 0.038 0.035 0.023
-
D A blood aample was removed from ment a.a a check for natural radioactivity. b This sample WBB lost in one of the ures.
3
s
0.000
0.000
0.008 -b 0.022 0.024 0.026 0.029 0.035 0.040 0.033
0.011 0.018 0.021 0.027 0.028 0.030 0.036 0.034 0.024
bird
(3) prior
concentration
to treatproced-
smallest concentration in the brain (Fig. 4). Nine tissues and/or organs had quantities of S35 within 0.006 rg per mg of the 48-hour blood sample. Table IV gives the mean uptake of S3” equivalents of CS,35 by 11 male and 14 female worms expelled from the treated birds 24 hours post-treatment. The mean amount of EY5recorded per worm was 0.087 pg per mg. Mean uptake of S35 for male and female worms was 0.1047 and 0.0821 (not significant) rg per mg, respectively. The amount of S35 per mg of excreta (urine and feces) is given in Table V. These data represent pg per mg of S35 for randomly selected samples rather than for each elimination, since many were passed within a few minutes of each other. The average yield of S35 per mg of excreta for all three chickens was 0.245 pg. The amount was two to three times greater than that recorded for the worms. Also, excreta containing worms was no different in S35 content, from excreta without worms. Since the fecal and urine components of the excreta were not separated prior to analysis, the fraction of S3j present in each could not be ascertained. McKee et al. (1943) indicated that in mammals 0.5% of the retained
cBRAIN FIG. 4. Comparison of S” equivalents of CSZW contained by Ascario!ia galli and certain tissues and organs of the host after treatment with the drug. Mean values are represented. (Gall bladder represents a cross section of frozen gall bladder and its contents.)
TABLE
IV
Mean ~9%Equivalents of CS:’ for Ascarids Expelled by Treated Chickens
Bird No.
Number worms
pg W per worm
pg W per mg of worm
7 12 6
8.01 10.15 8.75
0.076 0.091 0.093
1
2 3
TABLE
V
Mean S3’ Equivalents of CS? for Selected Sample of Excreta from Treated Chickens Bird
No. 1
2 3
pg W per mg of excreta 0.273 0.249 0.213
KNAPP, HANSEN, MOSER AND MCFARLAND
62
CSz3” occurred in the urine and none in the feces and that the remaining CS2 was accounted for as metabolites excreted in the urine. ACKNOWLEDGMENTS The authors wish to acknowledge the assistance of Dr. Stanley Wearden, Kansas State College Statistical Laboratory, and appreciation to the United States Atomic Energy Commission for funds supporting the study (Contract AT(ll-l)-
308). REFERENCES BRADY, F. J., LAWTON, A. H., COWIE, D. B., ANDREWS,H. L., NESS, A. T., ANDOGDEN,GLEN E. 1945. Localization of trivalent radioactive antimony following intravenous administration to dogs infected with Dirojiluria immitis. Am. J. Trop. Med. 25(2),
103-107.
DUNCAN, D. B. 1955. Multiple range and muItiple F tests. Biometrics 11, 142. EDWARDS,R. R., NESBETT,F. B., ANDSOLOMON, A. K. 1948. Recoil-activated and thermal exchange reactions between sulfur-35 and carbon disulfide. J. Am. Chem. Sot. 70,167O. ESSERMAN,H. B. 1952. The mode of action of phenothiazine as an anthelmintic. II. Phenothiazine in the intestinal fluid and nematode parasites of treated animals. Australian J. Sci. Research
B5(4), 485495.
FRITZ, J. S., ANDHAMMOND,G. S. 1957. Quantitative
Organic Analysis. John Wiley and Sons, Inc., New York, 303 pp. KNAPP, S. E., AND HANSEN, M. F. 1954. Observations on the anthelmintic action of carbon disulfide on the fowl ascarid Ascaridia galli (Schrank). J. Parasitol. 40, 17-18. LAWTON,A. H., NESS, A. T., BRADY,F. J., ANDDEAN, B. C. 1945. Distribution of radioactive arsenic following intraperitoneal injection of sodium arsenit.e into cotton rats infected with Litomosoides carinii. Science 102,121X122. LAZARUS,M. AND ROGERS,W. P. 1951. The mode of action of phenothiaeine as an anthelmintic. The uptake of ?S-labelled phenothiazine by the tissues of nematode parasites and their hosts. Australian J. Sci. Research B4(2), 163179. MCKEE, R. W., KIPER, C., FOUNTAIN,J. H., RISKIN, A. M., ANDDRINKER, P. 1943. A solvent vapor, carbon disulfide : Absorption, elimination, metabolism and mode of action. J. Am. Med. Assoc. 122,217-222.
PARR, S. W. 1919. A fusion bomb for sulfur determination in coal. J. Znd. Eng. Chem. 11,
230. TANG, P. C. 1938. The power function of the analysis of variance tests with tables and illustrations of their use. Stat. Research Mem. 2, 126157. TERHAAR, C. J. 1957. Carbon tetrachloride as an anthelmintic: Its effect on and distribution in a parasite (Ascaridia galli) and its host. Unpublished Dissertation, Kansas State College, 97 p.